This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. As biomacromolecules become more available, their applications in biomedicine are evident. Current efforts have focused on the utilization of such molecules either in their native state in solution or encapsulated in polymeric systems. Challenges such as protein stability in aqueous solutions, sensitivity to extreme changes in the surrounding environment, cost of production, low concentrations, the use of patient unsuited delivery systems, among others, have made the utilization of protein crystals an avenue to overcome such challenges. The encapsulation or confinement of such crystals in polymeric networks could potentially provide additional protection as well as the means to better control the dissolution of the crystal and the subsequent release of the protein. This project envisions the physicochemical understanding of the factors involved in protein crystal confinement in polymeric networks, which could be valuable for various applications including controlled drug delivery, biocatalysis, and biosensors. The main hypothesis is that crystal protein confinement in polymeric systems will significantly improve long term stability and release profiles of such biomacromolecules. Therefore, it encompasses the examination of the encapsulation of protein crystals in polymeric networks and their characterization. It is understood that crosslinked systems can provide not only protein protection, but additional release control as the dissolution rate will could be easily tailored to the necessary release rate. For this purpose, three types of crosslinked hydrogel systems will be examined, anionic, cationic, and neutral structures. This range of monomer chemical moieties will provide the means to examine polymer/crystal interactions as well as the means to control the release rate through the utilization of various crosslinker lengths and densities. These crossliked networks will be employed to encapsulate the following proposed proteins insulin, hemeproteins, and glucagon. These proteins present a wide range characteristics including isoelectric points and therefore their net charge will be different under the various polymerization conditions. Such results could be employed to relate surface charge with stability. As biomacromolecules become more available, their applications in biomedicine are evident. Current efforts have focused on the utilization of such molecules either in their native state in solution or encapsulated in polymeric systems. Challenges such as protein stability in aqueous solutions, sensitivity to extreme changes in the surrounding environment, cost of production, low concentrations, the use of patient unsuited delivery systems, among others, have made the utilization of protein crystals an avenue to overcome such challenges. The encapsulation or confinement of such crystals in polymeric networks could potentially provide additional protection as well as the means to better control the dissolution of the crystal and the subsequent release of the protein. This project envisions the physicochemical understanding of the factors involved in protein crystal confinement in polymeric networks, which could be valuable for various applications including controlled drug delivery, biocatalysis, and biosensors. The main hypothesis is that crystal protein confinement in polymeric systems will significantly improve long term stability and release profiles of such biomacromolecules. For this purpose, the following specific aims will be addressed: Specific Aim #1: Examination of the encapsulation of protein crystals in polymeric networks by (i) understanding the effects of polymer structure in protein crystal stability, (ii) the examination of the crystalline structure by x-ray diffraction, (iii) correlation of protein properties such as isoelectric point, molecular weight, crystalline structure, and outer shell protein charges with their stability in various polymer compositions. Hypothesis: Polymer morphology could be employed to maintain the stability of confined protein crystal structures. Specific Aim #2: Characterization of the proposed systems by the (i) examination of the protein release profiles and mechanisms, (ii) spectroscopic evaluation of protein structure after the crystal has dissolved via Raman, CD, and UV-VIS spectroscopy, and (iii) by the evaluation of protein biological activity and/or reactivity. Hypothesis: Protein crystal confinement in hydrogel networks will provide the means to control the dissolution rate of the protein crystal without affecting its biological activity/reactivity. Specific Aim #3: Specific Aim #3: Develop and implement computational methods to investigate the protein crystal confinement based on all atom Interparticle potentials. Hypothesis: By combining computational methods and experimental data a molecular level understanding of the structure, dynamics and stability of confined protein crystals would be obtained.